Aptamer-antibody on-chip sandwich immunoassay for detection of CRP in spiked serum.
This study describes a RNA aptamer-based biochip with high affinity and specificity for C-reactive protein (CRP). CRP, which exists in concentrations of 1-3mg/l in the serum of healthy patients, has been identified as a reliable biomarker for inflammation and as a potential marker for sepsis and tissue necrosis. The CRP-specific aptamer was covalently immobilized with its 5'-end on ARChip Epoxy. The detection of bound CRP was carried out optically using labelled secondary antibody in a sandwich format. Assay conditions were optimized with respect to the CRP binding buffer (buffer system, pH and additives) and Ca(2+) concentration (10 mM). Moreover, two sandwich immunoassay formats were tested, the one using dye-labelled antibodies and the other with biotin-modified antibodies/Dy647-labelled streptavidin. In comparison with an antibody-based chip assay, the aptamer chip is superior in terms of CRP measuring range (10 microg/l to 100mg/l) in human serum whereas antibody-based chips result in superior data reproducibility (CV of 8-15%). In contrast to antibody chips, aptamer microarrays provide the unique potential of detecting CRP in serum samples of low risk patients (1-3mg/l) as well as high risk patients (>500 mg/l), furthermore elevated CRP levels (20-350 mg/l) with acceptable recovery (70-130%) by including only one serum sample dilution step (1:100) for the complete measuring range.
Pultar
Johanna
J
Austrian Research Centers GmbH - ARC, Department of Bioresources, 2444 Seibersdorf, Austria.
Sauer
Ursula
U
Domnanich
Patrick
P
Preininger
Claudia
C
D000906:Antibodies / Q000737:chemistry*; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D001774:Blood Chemical Analysis / Q000295:instrumentation*; D002097:C-Reactive Protein / Q000032:analysis*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007118:Immunoassay / Q000295:instrumentation*; D056656:Lab-On-A-Chip Devices; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000906:Antibodies; D052157:Aptamers, Nucleotide; D002097:C-Reactive Protein